In vitro anti‐Trichomonas gallinae effects of Ziziphus vulgaris L. and Camellia sinensis (L.) Kuntze extracts

Abstract Background Trichomonas gallinae is a parasite that causes canker and severe loss and death, especially in young pigeons. Metronidazole (MTZ) is the recommended drug for treating avian trichomoniasis. Due to drug resistance, non‐chemical alternatives, such as medicinal plant extracts, are also considered possible therapies for this disease. Objectives This study compares the antitrichomonal effects of MTZ with extracts of Camellia sinensis and Ziziphus vulgaris on T. gallinae in vitro. Methods Samples of T. gallinae were taken from infected pigeons. Multi‐well plates with different concentrations (5, 10, 25, 50 and 100 µg/mL) of plant extracts were used for the in vitro study. Results The minimum inhibitory concentration (MIC) of C. sinensis extract was 25 µg/mL over 24 h, compared to 50 µg/mL for MTZ. The MIC value of the Z. vulgaris extracts was 50 µg/mL. Conclusions The results suggest that the extracts of Z. vulgaris and C. sinensis, as potential natural agents, could have anti‐avian trichomoniasis properties. This study also shows that MTZ, C. sinensis and Z. vulgaris are equally effective in preventing the growth of T. gallinae trophozoites in the culture.

Green tea, the dried leaf of the Camellia sinensis plant, contains a number of physiologically active substances, including polyphenols, methylxanthines, essential oils, proteins, vitamins and amino acids (Yamamoto et al., 1997).Previous research has focused on the Vet Med Sci.2024;10:e1432.
The most common ingredient in C. sinensis is catechins (Benelli et al., 2002).Tea catechins are primarily responsible for the biological benefits of C. sinensis, including lowering plasma lipid levels, reducing inflammation and having antibacterial, antiparasitic, anticancer and antioxidant properties (Bohm, 1998;Huang et al., 1998;Paveto et al., 2004;Vijaya et al., 1995).These chemicals, which belong to the flavan-3-ol family, have attracted a lot of interest lately due to their potential therapeutic properties.Among other health benefits, the powerful antioxidant and antiviral properties of catechins could help prevent disease (Katada et al., 2020;Sanlier et al., 2018;Yang et al., 2019).
According to the study by Paveto et al. (2004) on the anti-Trypanosoma cruzi activity of green tea in vitro, green tea catechins are effective therapeutics for Chagas disease.
Ziziphus vulgaris (jujube) belongs to the plant family Rhamnaceae.This plant is widely cultivated in Burma, Iran and several regions of India (Goli-malekabadi et al., 2014).According to phytochemical analysis, Z. vulgaris has been shown to contain bioactive substances, including cyclopeptide alkaloids.According to several studies, cyclopeptide alkaloids are the cause of the antiplasmodial and antimycobacterial abilities of Ziziphus.In addition, the plant has been found to contain terpenoids, alkaloids, steroids, polysaccharides and saponin glycosides (Dodangeh et al., 2017;Panseeta et al., 2011).Dodangeh et al. (2017) showed that Z. vulgaris extract induces programmed death in Acanthamoeba cysts and trophozoites in culture.Jujube is therefore considered a potential alternative medicinal plant for new antiparasitic drugs.
To our knowledge, no one has taken note of the antitrichomonal properties of C. sinensis and Z. vulgaris.So, the aim of this work is to compare the in vitro antitrichomonal effects of C. sinensis and Z. vulgaris against T. gallinae with those of MTZ.

Preparation of plant extract
The Z. vulgaris and C. sinensis plants were purchased from a Persian herbal market and confirmed by the Agriculture Faculty of Urmia University, Iran.The technique utilized by Baqer et al. (2014) underwent certain adjustments, and plant extracts were prepared.All the dry plant materials, leaves of green tea and dried fruits of jujube were put in an electrical blender (Moulinex) and turned into powder.Five hundred mL of 70% ethanol and 100 g of the powdered plant were combined with a magnetic stirrer and stirred for 2 h.The obtained solution was left intact at room temperature for 24 h and was filtered after stirring again.The solvent was removed using a rotary evaporator.For future applications, the semi-solid residual components were freeze-dried at 4 • C.

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS (Thermo Scientific) was used to examine the chemical composition of the extract.The split ratio for the helium-based carrier gas was 0.50 mL/min.The following GS conditions were met: initial 40 • C ramping to 250 • C, 80 • C per min for 3 min, with 250 • C injector and detector temperatures.Individual chemicals were identified by comparing their relative retention times on a capillary column to those of real samples and their peak-to-peak mass spectra to those obtained from authentic samples and published data (Khoshnejad et al., 2023).

Parasites
Twenty-five pigeons were purchased from a local breeder in Urmia, Iran.The pigeons were about 6-week old.T. gallinae was recovered using the wet mounting technique.Microbiological swabs moistened with the warm saline solution were used to collect samples from membrane lesions in the oropharyngeal region of the birds.Wet smears were prepared by rubbing the swabs on the glass slide.The approach of Samour and Naldo (2003) was used to confirm T. gallinae on the slides when magnified with a light microscope at 100× and 400× magnification.Flagellated and pear-shaped trophozoites could be seen under the microscope.T. gallinae trophozoites were extracted from the mouth (Seddiek et al., 2014).Diamond's trypticase, yeast extract and maltose (TYM) medium (Oxoid Ltd.) was used to prepare the parasite cultures (Tabari et al., 2017).The culture received 10% foetal calf serum (Sigma Chemical Co.) and was incubated at 37 • C.After 5 days, the cultures were examined to assess the growth of T. gallinae (Seddiek et al., 2014).
Penicillin and streptomycin (120 IU) were added in the initial phases to the subcultures; but antibiotic use was discontinued once axenic culture was achieved (Tabari et al., 2017).

In vitro assay
We performed the in vitro analysis using the Tabari et al. (2017) approach with some modifications.MTZ (Alborz Daru) was used as a conventional anti-T.gallinae drug.Dimethylsulfoxide (DMSO, 0.5%) was used to dilute MTZ and all plant extracts (Bharti et al., 2006;Malekifard et al., 2021).The response of T. gallinae to these substances was investigated by incubating the trophozoites with different concentrations of Z. vulgaris, C. sinensis extracts and MTZ in multi-well plates.
One hundred microliters of culture medium were added to each well.
Prediluted MTZ, Z. vulgaris and C. sinensis extracts, and 1 × 10 4 parasites were added to the culture to achieve final concentrations of 5, 10, 25, 50 and 100 μg/mL.The control was the plate with no treatment.
The wells were sealed by spreading Vaseline (50 μL) over them to create an anaerobic environment.The plates were then incubated for a further 72 h at a temperature of 37 • C (Tabari et al., 2017).Cultured trophozoites were counted with a hemocytometer.Trypan blue (0.4%) was added to the samples in equal parts to help distinguish between live and dead trophozoites (Tabari et al., 2017).At least three replicates of each experiment were used to obtain the results.The lowest concentration of a drug at which the parasites were no longer motile was called the minimum inhibitory concentration (MIC) (Seddiek et al., 2014).The following equation was used to calculate the growth inhibition (GI) percentage: In the above equation, A denotes the average number of trophozoites in the control group and B denotes the average number of trophozoites in the test group (Seddiek et al., 2014).

Statistical analysis
Statistical analysis was performed by SPSS (v.26.0).An analysis of variance examined the difference between the control and test groups.

In vitro results
Efficacy According to the study, C. sinensis was able to completely eliminate T.
gallinae in 48 h at the MIC of 25 μg/mL but took 72 h at the MIC of 10 μg/mL (Table 2).The control samples showed no decrease in cell populations.
Figure 1 shows the per cent GI% results for the groups treated with
Our research serves as evidence of the in vitro anti-T.gallinae activity of jujube extract.In this study, we identified catechin (22.67%) in the jujube extract.The jujube extract was found to contain small amounts of other compounds.The results obtained by earlier studies showed the antiparasitic qualities of catechin, quercitrin and Kaempferol, which were the main components of jujube in this study (Calzada, 2005;Dodson et al., 2011;Paveto et al., 2004).In addition, the results of this study support the results of previous investigations into the antiparasitic, antifungal and antimicrobial properties of jujube cyclopeptide alkaloids, tannins and saponins (Chung et al., 1998;Dodangeh et al., 2017;Panseeta et al., 2011).Our in vitro study showed that after adding the jujube extract (50 μg/mL) to the growth medium, trichomonads were no longer present after 24 h.Other studies have shown that jujube extract is effective in vitro against Acanthamoeba spp.and malaria (Panseeta et al., 2011).The amoebicidal effect of Z. vulgaris is related to its propensity to induce apoptosis, as Dodangeh et al.
reported.Previous research showed that inducing apoptosis in Z. vulgaris has a significant advantage over other widely used drugs, such as MTZ.The fact that Z. vulgaris has no cytotoxic effect on cultured macrophage cells, which is another benefit of the herb (Dodangeh et al., 2017).
In this study, caffeine (24.07%) was the main component of green tea extract, and the antiparasitic effect of this substance was reported in several studies.The results of these studies suggested that green tea extract and caffeine had anti-Acanthamoeba and antileishmanial effects (Hajihossein et al., 2020;Tadesse et al., 2015).According to previous research (Parvez et al., 2019), C. sinensis and its components showed antimicrobial activity against gram-positive and gram-negative multidrug-resistant bacteria.In addition, C. sinensis inhibited Leishmania amazonensis (dos Reis et al., 2013;Inacio et al., 2013), reduced exposure to Haemonchus contortus worms (Zhong et al., 2014), and inhibited promastigote and amastigote forms of Leishmania braziliensis (Inacio et al., 2014).In addition, Babesia spp., Eimeria spp.and T. cruzi were all inhibited by C. sinensis (Aboulaila et al., 2010;Jang et al., 2007;Paveto et al., 2004).The free-living birds (Tabari et al., 2017).These isolates have been found in a number of countries, including Spain, Belgium, the United States and Iran (Gerhold et al., 2008;Munoz et al., 1998;Rouffaer et al., 2014;Tabari et al., 2017).According to our research, Z. vulgaris and C. sinensis extracts can be more effective treatment options for the above isolates.
To assess MTZ resistance in T. gallinae strains (Rouffaer et al., 2014), a 24-h MIC breakpoint (15.6 μg/mL) was established.Our study revealed that the 24 h MIC of MTZ was 50 μg/mL, which differs significantly from that reported by Rouffaer et al., whose specified value differs.As a result, we were able to conclude that the T. gallinae strains used in this study exhibited high levels of MTZ resistance (Rouffaer et al., 2014).
Our results agreed with those of Tabari et al. (2017) and Malekifard et al. (2021), who confirmed the presence of MTZ-resistant strains of T. gallinae in Iran.

CONCLUSIONS
The results of this study suggest that Z.
± 0.22 a 7.68 ± 0.05 a 6.75 ± 0.35 a Note: Data are presented as mean ± SD. a-d Different superscript letters in a column indicate significant differences (p < 0.05).extracts was examined in vitro.To our knowledge, this is the first study of the anti-T.gallinae properties of these extracts.This study demonstrated the effectiveness of Z. vulgaris and C. sinensis extracts against T. gallinae.The results of our study agree with those of Malekifard et al. (2021), who found anti-T.gallinae effects of Zingiber officinale and Lavandula angustifolia alcoholic extracts, which were dose-and time-dependent.Another study by Tabari et al. demonstrated the antitrichomonal effect of Peganum harmala on T. gallinae.
results of the study by Fakae et al. showed that beers made from C. sinensis exhibit amoebic activity against Acanthamoeba trophozoites and were very successful in preventing the parasite from encysting.According to their research, C. sinensis could be a source of inhibitors of Acanthamoeba castellanii growth and encystation, which could be useful in the development of topical adjunctive treatments to supplement the chemotherapeutic drugs currently used to treat A. castellanii infections (Fakae et al., 2020).Avian trichomoniasis is commonly treated with MTZ; however, certain isolates of T. gallinae are resistant to the drugs, posing a risk to F I G U R E 1 Comparison of growth inhibition percentage (GI%) of Trichomonas gallinae trophozoites in the presence of different concentrations of Camellia sinensis, Ziziphus vulgar is extracts and metronidazole for 24 (A), 48 (B) and 72 (C) h.Different superscript letters in a column indicate significant differences (p < 0.05).
vulgaris and C. sinensis extracts could be used as potent natural anti-T.gallinae agents.Further research is needed to determine the potential adverse effects of Z. vulgaris and C. sinensis and to demonstrate their trichomonicidal properties in vivo.AUTHOR CONTRIBUTIONS Farnaz Malekifard, Behnam Rahimi and Bijan Esmaeilnejad contributed to conception, design, data collection, statistical analysis and drafting of the manuscript.Behnam Rahimi, Farnaz Malekifard and Bijan Esmaeilnejad contributed to conception, design, supervision of the study and drafting of the manuscript.All authors approved the final version for submission.
data for various concentrations of Z. vulgaris and C. sinensis extracts as anti-T.gallinaeagentsare presented in Table2.It could be observed that the extracts of Z. vulgaris and C. sinensis had antitrichomonal activity against T. gallinae in different concentrations and times.C. sinensis extract had a MIC of 25 μg/mL at 24 h compared to 50 μg/mL for MTZ.For Z. vulgaris, these doses were 50 and 10 μg/mL in 48 and 72 h, respectively.It is interesting to note that the MIC for MTZ was determined to be 25 and 10 μg/mL at 48 and 72 h, respectively.
Major chemical compounds in Ziziphus vulgaris and Camellia sinensis extract identified by gas chromatography-mass spectrometry (GC-MS).
Z. vulgaris and C. sinensis extracts and MTZ for 24, 48 and 72 h.The data reveals a significant difference in GI% between the groups treated with Z. vulgaris and C. sinensis extracts and MTZ as compared to the control group.According to Figure1, the GI% of Z. vulgaris and C. sinen-TA B L E 1extracts in all doses used was also similar to that of MTZ.Therefore, the study shows that Z. vulgaris and C. sinensis extracts have a potent anti-T.gallinae effect in vitro.
Effect of various metronidazole, Ziziphus vulgaris and Camellia sinensis extract concentrations on the in vitro growth of Trichomonas gallinae trophozoites (10 4 ).
TA B L E 2